Novel Bacillus thuringiensis isolate denoted B.t. PS81F, active against lepidopteran pests, and a gene encoding a lepidopteran-active toxin

ABSTRACT

A novel B.t. toxin gene encoding a protein toxic to lepidopteran insects has been cloned from a novel lepidopteran-active B. thuringiensis microbe. The DNA encoding the B.t. toxin can be used to transform various prokaryotic and eukaryotic microbes to express the B.t. toxin. These recombinant microbes can be used to control lepidopteran insects in various environments.

This is a division of application Ser. No. 07/263,567, filed Oct. 27, 1988, now abandoned.

BACKGROUND OF THE INVENTION

The most widely used microbial pesticides are derived from the bacterium Bacillus thuringiensis. This bacterial agent is used to control a wide range of leaf-eating caterpillars, and mosquitos. Bacillus thuringiensis produces a proteinaceous paraspore or crystal which is toxic upon ingestion by a susceptible insect host. For example, B. thuringiensis var. kurstaki HD-1 produces a crystal called a delta toxin which is toxic to the larvae of a number of lepidopteran insects. The cloning and expression of this B.t. crystal protein gene in Escherichia coli has been described in the published literature (Schnepf, H.E. and Whitely, H.R. [1981] Proc. Natl. Acad. Sci. U.S.A. 78:2893-2897). U.S. Pat. No. 4,448,885 and U.S. Pat. No. 4,467,036 both disclose the expression of B.t. crystal protein in E. coli.

BRIEF SUMMARY OF THE INVENTION

The subject invention concerns a novel Bacillus thuringiensis isolate designated B.t. PS81F which has activity against all lepidopteran pests tested.

Also disclosed and claimed is a novel toxin gene toxic to lepidopteran insects. This toxin gene can be transferred to suitable hosts via a plasmid vector.

Specifically, the invention comprises a novel B.t. isolate denoted B.t. PS81F, mutants thereof, and a novel delta endotoxin gene which encodes a 133,266 dalton protein which is active against lepidopteran pests.

Table 1 discloses the DNA encoding the novel toxin. Table 2 discloses the amino acid sequence of the novel toxin. Table 3 is a composite of Tables 1 and 2. Table 4 shows a comparison of the deduced amino acid sequence of 81F with five other known B.t. endotoxins.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1: Agarose gel electrophoresis of plasmid preparations from B.t. PS81F and B.t. HO-1.

DETAILED DISCLOSURE OF THE INVENTION

The novel toxin gene of the subject invention was obtained from a novel lepidopteran-active B. thuringiensis (B.t.) isolate designated PS81F.

Characteristics of B.t. PS81F

Colony morphology--Large colony, dull surface, typical B.t.

Vegetative cell morphology--typical B.t.

Flagellar serotype--4a4c, kenya.

Intracellular inclusions--sporulating cells produce a bipyramidal crystal.

Plasmid preparations--agarose gel electrophoresis of plasmid preparations distinguishes B.t. PS81F from B.t. HD-1 and other B.t. isolates.

Alkali-soluble proteins--B.t. PS81F has a 130,000 dalton protein and a 60,000 dalton protein.

Activity--B.t. PS81F kills all Lepidoptera tested.

    ______________________________________                                         Bioassay results:     LC50                                                     ______________________________________                                         Beet armyworm, Spodoptera exigua                                                                     10.4 ug/ml                                               Western spruce budworm,                                                                               1.4 ug/ml                                               Choristoneura occidentalis                                                     ______________________________________                                    

Bioassay procedures:

Spodoptera exigua--dilutions are prepared of a spore and crystal pellet, mixed with USDA Insect Diet (Technical Bulletin 1528, U.S. Department of Agriculture) and poured into small plastic trays. Neonate Spodoptera exigua larvae are placed on the diet mixture and held at 25° C. Mortality is recorded after six days.

Choristoneura occidentalis--dilutions and diet are prepared in the same manner as for the Spodoptera exigua bioassay. Fourth instar larvae are used, and mortality is recorded after eight days.

B. thuringiensis PS81F, NRRL B-18424, and mutants thereof, can be cultured using standard known media and fermentation techniques. Upon completion of the fermentation cycle, the bacteria can be harvested by first separating the B.t. spores and crystals from the fermentation broth by means well known in the art. The recovered B.t. spores and crystals can be formulated into a wettable powder, a liquid concentrate, granules or other formulations by the addition of surfactants, dispersants, inert carriers and other components to facilitate handling and application for particular target pests. The formulation and application procedures are all well known in the art and are used with commercial strains of B. thuringiensis (HD-1) active against Lepidoptera, e.g., caterpillars. B.t. PS81F, and mutants thereof, can be used to control lepidopteran pests.

A subculture of B.t. PS81F and the E. coli host harboring the toxin gene of the invention, E. coli DH5(α), containing the plasmid pMYC386, was deposited in the permanent collection of the Northern Research Laboratory, U.S. Department of Agriculture, Peoria, Ill., U.S.A. on Oct. 7, 1988. The accession numbers are as follows:

B.t. PS81F--NRRL B-18424

E. coli (DH5α) (pMYC386)--NRRL B-18423

The subject cultures have been deposited under conditions that assure that access to the cultures will be available during the pendency of this patent application to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 CFR 1.14 and 35 USC 122. The deposits are available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny, are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.

Further, the subject culture deposits will be stored and made available to the public in accord with the provisions of the Budapest Treaty for the Deposit of Microorganisms, i.e., they will be stored with all the care necessary to keep them viable and uncontaminated for a period of at least five years after the most recent request for the furnishing of a sample of the deposit, and in any case, for a period of at least 30 (thirty) years after the date of deposit or for the enforceable life of any patent which may issue disclosing the cultures. The depositor acknowledges the duty to replace the deposits should the depository be unable to furnish a sample when requested, due to the condition of the deposit(s). All restrictions on the availability to the public of the subject culture deposits will be irrevocably removed upon the granting of a patent disclosing them.

The toxin gene of the subject invention can be introduced into a wide variety of microbial hosts. Expression of the toxin gene results. directly or indirectly, in the intracellular production and maintenance of the pesticide. With suitable hosts, e.g., Pseudomonas, the microbes can be applied to the situs of lepidopteran insects where they will proliferate and be ingested by the insects. The result is a control of the unwanted insects. Alternatively, the microbe hosting the toxin gene can be treated under conditions that prolong the activity of the toxin produced in the cell. The treated cell then can be applied to the environment of target pest(s). The resulting product retains the toxicity of the B.t. toxin.

Where the B.t. toxin gene is introduced via a suitable vector into a microbial host, and said host is applied to the environment in a living state, it is essential that certain host microbes be used. Microorganism hosts are selected which are known to occupy the "phytosphere" (phylloplane, phyllosphere, rhizosphere, and/or rhizoplane) of one or more crops of interest. These microorganisms are selected so as to be capable of successfully competing in the particular environment (crop and other insect habitats) with the wild-type microorganisms, provide for stable maintenance and expression of the gene expressing the polypeptide pesticide, and, desirably, provide for improved protection of the pesticide from environmental degradation and inactivation.

A large number of microorganisms are known to inhabit the phylloplane (the surface of the plant leaves) and/or the rhizosphere (the soil surrounding plant roots) of a wide variety of important crops. These microorganisms include bacteria, algae, and fungi. Of particular interest are microorganisms, such as bacteria, e.g., genera Pseudomonas, Erwinia Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas, Methylius, AGrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes; fungi, particularly yeast, e.g., genera Saccharomyces, Cryptococcus, Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium. Of particular interest are such phytosphere bacterial species as Pseudomonas syringae. Pseudomonas fluorescens, Serratia marcescens, Acetobacter xylinum, Agrobacteria, Rhodopseudomonas spheroides, Xanthomonas campestris, Rhizobium melioti, Alcaligenes entrophus, and Azotobacter vinlandii; and phytosphere yeast species such as Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca, Cryptococcus albidus, C. diffluens, C. laurentii, Saccharomyces rosei, S. pretoriensis, S. cerevisiae, Sporobolomyces roseus, S. odorus, Kluyveromyces veronae, and Aureobasidium pollulans. Of particular interest are the pigmented microorganisms.

A wide variety of ways are available for introducing the B.t. gene expressing the toxin into the microorganism host under conditions which allow for stable maintenance and expression of the gene. One can provide for DNA constructs which include the transcriptional and translational regulatory signals for expression of the toxin gene, the toxin gene under their regulatory control and a DNA sequence homologous with a sequence in the host organism, whereby integration will occur, and/or a replication system which is functional in the host, whereby integration or stable maintenance will occur.

The transcriptional initiation signals will include a promoter and a transcriptional initiation start site. In some instances, it may be desirable to provide for regulative expression of the toxin, where expression of the toxin will only occur after release into the environment. This can be achieved with operators or a region binding to an activator or enhancers, which are capable of induction upon a change in the physical or chemical environment of the microorganisms. For example, a temperature sensitive regulatory region may be employed, where the organisms may be grown up in the laboratory without expression of a toxin, but upon release into the environment, expression would begin. Other techniques may employ a specific nutrient medium in the laboratory, which inhibits the expression of the toxin, where the nutrient medium in the environment would allow for expression of the toxin. For translational initiation, a ribosomal binding site and an initiation codon will be present.

Various manipulations may be employed for enhancing the expression of the messenger, particularly by using an active promoter, as well as by employing sequences, which enhance the stability of the messenger RNA. The initiation and translational termination region will involve stop codon(s), a terminator region, and optionally, a polyadenylation signal.

In the direction of transcription, namely in the 5' to 3' direction of the coding or sense sequence, the construct will involve the transcriptional regulatory region, if any, and the promoter, where the regulatory region may be either 5' or 3' of the promoter, the ribosomal binding site, the initiation codon, the structural gene having an open reading frame in phase with the initiation codon, the stop codon(s), the polyadenylation signal sequence, if any, and the terminator region. This sequence as a double strand may be used by itself for transformation of a microorganism host, but will usually be included with a DNA sequence involving a marker, where the second DNA sequence may be joined to the toxin expression construct during introduction of the DNA into the host.

By a marker is intended a structural gene which provides for selection of those hosts which have been modified or transformed. The marker will normally provide for selective advantage, for example, providing for biocide resistance, e.g., resistance to antibiotics or heavy metals; complementation, so as to provide prototropy to an auxotrophic host, or the like. Preferably, complementation is employed, so that the modified host may not only be selected, but may also be competitive in the field. One or more markers may be employed in the development of the constructs, as well as for modifying the host. The organisms may be further modified by providing for a competitive advantage against other wild-type microorganisms in the field. For example, genes expressing metal chelating agents, e.g., siderophores, may be introduced into the host along with the structural gene expressing the toxin. In this manner, the enhanced expression of a siderophore may provide for a competitive advantage for the toxin-producing host, so that it may effectively compete with the wild-type microorganisms and stably occupy a niche in the environment.

Where no functional replication system is present, the construct will also include a sequence of at least 50 basepairs (bp), preferably at least about 100 bp, and usually not more than about 1000 bp of a sequence homologous with a sequence in the host. In this way, the probability of legitimate recombination is enhanced, so that the gene will be integrated into the host and stably maintained by the host. Desirably, the toxin gene will be in close proximity to the gene providing for complementation as well as the gene providing for the competitive advantage. Therefore, in the event that a toxin gene is lost, the resulting organism will be likely to also lose the complementing gene and/or the gene providing for the competitive advantage, so that it will be unable to compete in the environment with the gene retaining the intact construct.

A large number of transcriptional regulatory regions are available from a wide variety of microorganism hosts, such as bacteria, bacteriophage, cyanobacteria, algae, fungi, and the like. Various transcriptional regulatory regions include the regions associated with the trp gene, lac gene, gal gene, the lambda left and right promoters, the Tac promoter, the naturally-occurring promoters associated with the toxin gene, where functional in the host. See for example, U.S. Pat. Nos. 4,332,898, 4,342,832 and 4,356,270. The termination region may be the termination region normally associated with the transcriptional initiation region or a different transcriptional initiation region, so long as the two regions are compatible and functional in the host.

Where stable episomal maintenance or integration is desired, a plasmid will be employed which has a replication system which is functional in the host. The replication system may be derived from the chromosome, an episomal element normally present in the host or a different host, or a replication system from a virus which is stable in the host. A large number of plasmids are available, such as pBR322, pACYC184, RSFIOIO, pR01614, and the like. See for example, Olson et al., (1982) J. Bacteriol. 150:6069, and Bagdasarian et al., (1981) Gene 16:237, and U.S. Pat. Nos. 4,356,270, 4,362,817, and 4,371,625.

The B.t. gene can be introduced between the transcriptional and translational initiation region and the transcriptional and translational termination region, so as to be under the regulatory control of the initiation region. This construct will be included in a plasmid, which will include at least one replication system, but may include more than one, where one replication system is employed for cloning during the development of the plasmid and the second replication system is necessary for functioning in the ultimate host. In addition, one or more markers may be present, which have been described previously. Where integration is desired, the plasmid will desirably include a sequence homologous with the host genome.

The transformants can be isolated in accordance with conventional ways, usually employing a selection technique, which allows for selection of the desired organism as against unmodified organisms or transferring organisms, when present. The transformants then can be tested for pesticidal activity.

Suitable host cells, where the pesticide-containing cells will be treated to prolong the activity of the toxin in the cell when the then treated cell is applied to the environment of target pest(s), may include either prokaryotes or eukaryotes, normally being limited to those cells which do not produce substances toxic to higher organisms, such as mammals. However, organisms which produce substances toxic to higher organisms could be used, where the toxin is unstable or the level of application sufficiently low as to avoid any possibility of toxicity to a mammalian host. As hosts, of particular interest will be the prokaryotes and the lower eukaryotes, such as fungi. Illustrative prokaryotes, both Gram-negative and -positive, include Enterobacteriaceae, such as Escherichia, Erwinia, Shigella, Salmonella, and Proteus; Bacillaceae; Rhizobiceae, such as Rhizobium; Spirillaceae, such as photobacterium, Zymomonas, Serratia, Aeromonas, Vibrio, Desulfovibrio, Spirillum; Lactobacillaceae; Pseudomonadaceae, such as Pseudomonas and Acetobacter; Azotobacteraceae and Nitrobacteraceae. Among eukaryotes are fungi, such as Phycomycetes and Ascomycetes, which includes yeast, such as Saccharomyces and Schizosaccharomyces; and Basidiomycetes yeast, such as Rhodotorula, Aureobasidium, Sporobolomyces, and the like.

Characteristics of particular interest in selecting a host cell for purposes of production include ease of introducing the B.t. gene into the host, availability of expression systems, efficiency of expression, stability of the pesticide in the host, and the presence of auxiliary genetic capabilities. Characteristics of interest for use as a pesticide microcapsule include protective qualities for the pesticide, such as thick cell walls, pigmentation, and intracellular packaging or formation of inclusion bodies; leaf affinity; lack of mammalian toxicity; attractiveness to pests for ingestion; ease of killing and fixing without damage to the toxin; and the like. Other considerations include ease of formulation and handling, economics, storage stability, and the like.

Host organisms of particular interest include yeast, such as Rhodotorula sp., Aureobasidium sp., Saccharomyces sp., and Sporobolomyces sp.; phylloplane organisms such as Pseudomonas sp., Erwinia sp. and Flavobacterium sp.; or such other organisms as Escherichia, Lactobacillus sp., Bacillus sp., and the like. Specific organisms include Pseudomonas aeruginosa, Pseudomonas fluorescens, Saccharomyces cerevisiae, Bacillus thuringiensis, Escherichia coli, Bacillus subtilis, and the like.

The cell will usually be intact and be substantially in the proliferative form when treated, rather than in a spore form, although in some instances spores may be employed.

Treatment of the microbial cell, e.g., a microbe containing the B.t. toxin gene, can be by chemical or physical means, or by a combination of chemical and/or physical means, so long as the technique does not deleteriously affect the properties of the toxin, nor diminish the cellular capability in protecting the toxin. Examples of chemical reagents are halogenating agents, particularly halogens of atomic no. 17-80. More particularly, iodine can be used under mild conditions and for sufficient time to achieve the desired results. Other suitable techniques include treatment with aldehydes, such as formaldehyde and glutaraldehyde; anti-infectives, such as zephiran chloride and cetylpyridinium chloride; alcohols, such as i sopropyl and ethanol; various histologic fixatives, such as Bouin's fixative and Helly's fixative (See: Humason, Gretchen L., Animal Tissue Techniques, W.H. Freeman and Company, 1967); or a combination of physical (heat) and chemical agents that preserve and prolong the activity of the toxin produced in the cell when the cell is administered to the host animal. Examples of physical means are short wavelength radiation such as gamma-radiation and X-radiation, freezing, UV irradiation, lyophilization, and the like.

The cells generally will have enhanced structural stability which will enhance resistance to environmental conditions. Where the pesticide is in a proform, the method of inactivation should be selected so as not to inhibit processing of the proform to the mature form of the pesticide by the target pest pathogen. For example, formaldehyde will crosslink proteins and could inhibit processing of the proform of a polypeptide pesticide. The method of inactivation or killing retains at least a substantial portion of the bio-availability or bioactivity of the toxin.

The cellular host containing the B.t. insecticidal gene may be grown in any convenient nutrient medium, where the DNA construct provides a selective advantage, providing for a selective medium so that substantially all or all of the cells retain the B.t. gene. These cells may then be harvested in accordance with conventional ways. Alternatively, the cells can be treated prior to harvesting.

The B.t. cells may be formulated in a variety of ways. They may be employed as wettable powders, granules or dusts, by mixing with various inert materials, such as inorganic minerals (phyllosilicates, carbonates, sulfates, phosphates, and the like) or botanical materials (powdered corncobs, rice hulls, walnut shells, and the like). The formulations may include spreader-sticker adjuvants, stabilizing agents, other pesticidal additives, or surfactants. Liquid formulations may be aqueous-based or non-aqueous and employed as foams, gels, suspensions, emulsifiable concentrates, or the like. The ingredients may include rheological agents, surfactants, emulsifiers, dispersants, or polymers.

The pesticidal concentration will vary widely depending upon the nature of the particular formulation, particularly whether it is a concentrate or to be used directly. The pesticide will be present in at least 1% by weight and may be 100% by weight. The dry formulations will have from about 1-95% by weight of the pesticide while the liquid formulations will generally be from about 1-60% by weight of the solids in the liquid phase. The formulations will generally have from about 10² to about 10⁴ cells/mg. These formulations will be administered at about 50 mg (liquid or dry) to 1 kg or more per hectare.

The formulations can be applied to the environment of the lepidopteran pest(s), e.g., plants, soil or water, by spraying, dusting, sprinkling, or the like.

Mutants of PS81F can be made by procedures well known in the art. For example, an asporogenous mutant can be obtained through ethylmethane sulfonate (EMS) mutagenesis of PS81F. The mutants can be made using ultraviolet light and nitrosoguanidine by procedures well known in the art.

Following are examples which illustrate procedures, including the best mode, for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

EXAMPLE 1 CULTURING B.t. PS81F, NRRL B-18424

A subculture of B.t. PS81F, NRRL B-18424, or mutants thereof, can be used to inoculate the following medium, a peptone, glucose, salts medium.

    ______________________________________                                         Bacto Peptone          7.5    g/l                                              Glucose                1.0    g/l                                              KH.sub.2 PO.sub.4      3.4    g/l                                              K.sub.2 HPO.sub.4      4.35   g/l                                              Salt Solution          5.0    ml/l                                             CaCl.sub.2 Solution    5.0    ml/l                                             Salts Solution (100 ml)                                                        MgSO.sub.4.7H.sub.2 O  2.46   g                                                MnSO.sub.4.H.sub.2 O   0.04   g                                                ZnSO.sub.4.7H.sub.2 O  0.28   g                                                FeSO.sub.4.7H.sub.2 O  0.40   g                                                CaCl.sub.2 Solution (100 ml)                                                   CaCl.sub.2.2H.sub.2 O  3.66   g                                                pH 7.2                                                                         ______________________________________                                    

The salts solution and CaCl₂ solution are filter-sterilized and added to the autoclaved and cooked broth at the time of inoculation. Flasks are incubated at 30° C. on a rotary shaker at 200 rpm for 64 hr.

The above procedure can be readily scaled up to large fermentors by procedures well known in the art.

The B.t. spores and/or crystals, obtained in the above fermentation, can be isolated by procedures well known in the art. A frequently-used procedure is to subject the harvested fermentation broth to separation techniques, e.g., centrifugation.

EXAMPLE 2 Cloning of Novel Toxin Gene and Transformation into Escherichia coli

Total cellular DNA was prepared by growing the cells of B. thuringiensis HD-1 and the novel B.t. PS81F to a low optical density (OD₆₀₀ =1.0) and recovering the cells by centrifugation. The cells were protoplasted in TES buffer (30 mM Tris-Cl, 10 mM EDTA, 50 mM NaCl, pH=8.0) containing 20 % sucrose and 50 mg/ml lysozyme. The protoplasts were lysed by addition of SDS to a final concentration of 4%. The cellular material was precipitated overnight at 4° C. in 100 mM final concentration neutral potassium chloride. The supernate was extracted twice with phenol/chloroform (1:1). The DNA was precipitated in ethanol and purified by isopycnic banding on a cesium chloride gradient.

Total cellular DNA from each (PS81F and HD-1) was digested with EcoRI and separated by electrophoresis on a 0.8% Agarose-TAE-buffered gel. A Southern blot of the gel was probed with the NsiI to NsiI fragment of the toxin gene contained in plasmid pM3,130-7 of NRRL B-18332 and the NsiI to KpnI fragment of the "4.5 Kb class" toxin gene (Kronstad and Whitely [1986] Gene USA 43:29-40). These two fragments were combined and used as the probe. Results show that hybridizing fragments of PS81F are distinct from those of HD-1. Specifically, a 3.5 Kb hybridizing band in PS81F was detected instead of the 300 bp larger 3.8 Kb hybridizing band seen in HD-1.

Two hundred micrograms of PS81F total cellular DNA was digested with EcoRI and separated by electrophoresis on a preparative 0.8% Agarose-TAE gel. The 3.0 Kb to 4.0 Kb region of the gel was cut out and the DNA from it was electroeluted and concentrated using an ELUTIP™-d (Schleicher and Schuell, Keene, NH) ion exchange column. The isolated EcoRI fragments were ligated to LAMBDA ZAP™ EcoRI arms (Stratagene Cloning Systems, La Jolla, CA) and packaged packaged using GIGAPACK GOLD™ extracts. The packaged recombinant phage were plated with E. coli strain BB4 (Stratagene) to give high plaque density. The plaques were screened by standard nucleic acid hybridization procedure with radiolabeled probe. The plaques that hybridized were purified and re-screened at a lower plaque density. The resulting purified phage were grown with R408 M13 helper phage (Stratagene) and the recombinant BLUESCRIPT™ (Stratagene) plasmid was automatically excised and packaged. The "phagemid" was re-infected in XLl-Blue E. coli cells (Stratagene) as part of the automatic excision process. The infected XLl-Blue cells were screened for ampicillin resistance and the resulting colonies were analyzed by standard miniprep procedure to find the desired plasmid. The plasmid, designated pM5,31-1, contained an approximate 3.5 Kb EcoRI insert and was sequenced using Stratagene's T7 and T3 primers plus a set of existing B.t. endotoxin oligonucleotide primers. About 1.7 Kb of the toxin gene was sequenced and data analysis comparing PS81F to other cloned B.t. endotoxin genes showed that the PS81F sequence was unique. A synthetic oligonucleotide (GCTGAAGAACTTCCTATTCGTGGTGGTGAGC) was constructed to one of the regions in the PS81F sequence that was least homologous relative to other existing B.t. endotoxin genes.

Total cellular DNA partially digested with Sau3A and fractionated by electrophoresis into a mixture of 9-23 Kb fragments on a 0.6% agarose TAE gel was ligated into LAMBDA DASH™ (Stratagene). The packaged phage were plated out with P2392 E. coli cells (Stratagene) at a high titer and screened using the radiolabeled synthetic oligonucleotide supra as a nucleic acid hybridization probe. Hybridizing plaques were rescreened at a lower plaque density. A purified hybridizing plaque was used to infect P2392 E. coli cells in liquid culture for preparation of phage for DNA isolation. DNA was isolated by standard procedures. Preparative amounts of recombinant phage DNA were digested with SalI (to release the inserted DNA from lambda arms) and separated by electrophoresis on a 0.6% Agarose-TAE gel. The large fragments (electroeluted and concentrated as described above) were ligated to an XhoI digested and phosphatased BLUESCRIPT™ plasmid. The ligation was transformed into E. coli DH5(α) competent cells (BRL) and plated on LB agar containing ampioillin, isopropyl-(β)-D-thiogalactoside (IPTG) and 5-bromo-4-chloro-3-indolyl-(β)-D-galactoside (XGAL). White colonies (with insertions in the (β)-galactosidase gene of pBluescript) were subjected to standard miniprep procedures to isolate the plasmid, designated pMI,43-24. The full length toxin gene was sequenced by using oligonucleotide primers made to the "4.3 Kb class" toxin gene and by "walking" with primers made to the sequence of PS81F. Data analysis comparing the deduced PS81F amino acid sequence to the sequences of five other endotoxins shows PS81F to be unique (Table 4).

The plasmid pM1,43-24 contains about 18 Kb of PS81F DNA including the 3.518 Kb which codes for the 133,266 dalton endotoxin. The plasmid was reduced in size by cutting out approximately 13 Kb of non-coding DNA, ligating the ends, transforming DH5(β) and plating on LB agar containing ampicillin. The resulting colonies were analyzed by standard miniprep procedures to isolate plasmids that were reduced in size. The desired plasmid, pMYC386, contains the coding sequence of the PS81F toxin gene, which could be excised as an SaeI to ApaI 4.5 Kb fragment.

The above cloning procedures were conducted using standard procedures unless otherwise noted.

The various methods employed in the preparation of the plasmids and transformation of host organisms are well known in the art. Also, methods for the use of lambda bacteriophage as a cloning. vehicle, i.e., the preparation of lambda DNA, in vitro packaging, and transfection of recombinant DNA, are well known in the art. These procedures are all described in Maniatis, T., Fritsch, E.F., and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y. Thus, it is within the skill of those in the genetic engineering art to extract DNA from microbial cells, perform restriction enzyme digestions, electrophorese DNA fragments, tail and anneal plasmid and insert DNA, ligate DNA, transform cells, prepare plasmid DNA, electrophorese proteins, and sequence DNA.

The restriction enzymes disclosed herein can be purchased from Bethesda Research Laboratories, Gaithersburg, Md., or New England Biolabs, Beverly, Mass. The enzymes are used according to the instructions provided by the supplier.

Plasmid pMYC386 containing the B.t. toxin gene, can be removed from the transformed host microbe by use of standard well-known procedures. For example, E. coli NRRL B-1 8423 can be subjected to cleared lysate isopycnic density gradient procedures, and the like, to recover pMYC386.

Data from standard insect tests show that novel B.t. PS81F is active against diamondback moth, Spodoptera exigua, Western spruce budworm, and T. ni.

EXAMPLE 3 Insertion of Toxin Gene Into Plants

The novel gene coding for the novel insecticidal toxin, as disclosed herein, can be inserted into plant cells using the Ti plasmid from Agrobacter tumefaciens. Plant cells can then be caused to regenerate into plants (Zambryski, P., Joos, H., Gentello, C., Leemans, J., Van

Montague, M. and Schell, J [1983] Cell 32:1033-1043). A particularly useful vector in this regard is pEND4K (Klee, H.J., Yanofsky, M.F. and Nester, E.W. [1985] Bio/Technology 3:637-642). This plasmid can replicate both in plant cells and in bacteria and has multiple cloning sites for passenger genes. The toxin gene, for example, can be inserted into the BamHI site of pEND4K, propagated in E. coli, and transformed into appropriate plant cells.

EXAMPLE 4 Cloning of Novel B. thuringiensis Gene Into Baculoviruses

The novel gene of the invention can be cloned into baculoviruses such as Autographa californica nuclear polyhedrosis virus (AcNPV). Plasmids can be constructed that contain the AcNPV genome cloned into a commercial cloning vector such as pUC8. The AcNPV genome is modified so that the coding region of the polyhedrin gene is removed and a unique cloning site for a passenger gene is placed directly behind the polyhedrin promoter. Examples of such vectors are pGP-B6874, described by Pennock et al. (Pennock, G.D., Shoemaker, C. and Miller, L.K. [1984] Mol. Cell. Biol. 4:399-406), and pAC380, described by Smith et al. (Smith, G.E., Summers, M.D. and Fraser, M.J. [1983] Mol Cell. Biol. 3:2156-2165). The gene coding for the novel protein toxin of the invention can be modified with BamHI linkers at appropriate regions both upstream and downstream from the coding region and inserted into the passenger site of one of the AcNPV vectors.

As disclosed previously, the nucleotide sequence encoding the novel B.t. toxin gene is shown in Table 1. The deduced amino acid sequence is shown in Table 2.

It is well known in the art that the amino acid sequence of a protein is determined by the nucleotide sequence of the DNA. Because of the redundancy of the genetic code, i.e., more than one coding nucleotide triplet (codon) can be used for most of the amino acids used to make proteins, different nucleotide sequences can code for a particular amino acid. Thus, the genetic code can be depicted as follows:

    ______________________________________                                         Phenylalanine (Phe)                                                                          TTK      Histidine (His)                                                                              CAK                                       Leucine (Leu) XTY      Glutamine (Gln)                                                                              CAJ                                       Isoleucine (Ile)                                                                             ATM      Asparagine (Asn)                                                                             AAK                                       Methionine (Met)                                                                             ATG      Lysine (Lys)  AAJ                                       Valine (Val)  GTL      Aspartic acid (Asp)                                                                          GAK                                       Serine (Ser)  QRS      Glutamic acid (Glu)                                                                          GAJ                                       Proline (Pro) CCL      Cysteine (Cys)                                                                               TGK                                       Threonine (Thr)                                                                              ACL      Tryptophan (Trp)                                                                             TGG                                       Alanine (Ala) GCL      Arginine (Arg)                                                                               WGZ                                       Tyrosine (Tyr)                                                                               TAK      Glycine (Gly) GGL                                       Termination signal                                                                           TAJ                                                              ______________________________________                                    

Key: Each 3-letter deoxynucleotide triplet corresponds to a trinucleotide of mRNA, having a 5'-end on the left and a 3'-end on the right. All DNA sequences given herein are those of the strand whose sequence correspond to the mRNA sequence, with thymine substituted for uracil. The letters stand for the purine or pyrimidine bases forming the deoxynucleotide sequence.

A=adenine

G=guanine

C=cytosine

T=thymine

X=T or C if Y is A or G

X=C if Y is C or T

Y=A, G, C or T if X is C

Y=A or G if X is T

W=C or A if Z is A or G

W=C if Z is C or T

Z=A, G, C or T if W is C

Z=A or G if W is A

QR=TC if S is A, G, C or T; alternatively QR=AG if S is T or C

J=A or G

K=T or C

L=A, T, C or G

M=A, C or T

The above shows that the novel amino acid sequence of the B.t. toxin can be prepared by equivalent nucleotide sequences encoding the same amino acid sequence of the protein. Accordingly, the subject invention includes such equivalent nucleotide sequences. In addition it has been shown that proteins of identified structure and function may be constructed by changing the amino acid sequence if such changes do not alter the protein secondary structure (Kaiser, E.T. and Kezdy, F.J. [1984] Science 223:249-255). Thus, the subject invention includes mutants of the amino acid sequence depicted herein which do not alter the protein secondary structure, or if the structure is altered, the biological activity is retained to some degree.

                                      TABLE 1                                      __________________________________________________________________________     Nucleotide sequence of novel toxin encoding gene.                              __________________________________________________________________________        10        20        30        40        50        60                        1  ATGGAGATAG                                                                               TGAATAATCA                                                                               GAATCAATGC                                                                               GTGCCTTATA                                                                               ATTGTTTAAA                                                                               TAATCCTGAA                61 AATGAGATAT                                                                               TAGATATTGA                                                                               AAGGTCAAAT                                                                               AGTACTGTAG                                                                               CAACAAACAT                                                                               CGCCTTGGAG                121                                                                               ATTAGTCGTC                                                                               TGCTCGCTTC                                                                               CGCAACTCCA                                                                               ATAGGGGGGA                                                                               TTTTATTAGG                                                                               ATTGTTTGAT                181                                                                               GCAATATGGG                                                                               GGTCTATAGG                                                                               CCCTTCACAA                                                                               TGGGATTTAT                                                                               TTTTAGAGCA                                                                               AATTGAGCTA                241                                                                               TTGATTGACC                                                                               AAAAAATAGA                                                                               GGAATTCGCT                                                                               AGAAACCAGG                                                                               CAATTTCTAG                                                                               ATTAGAAGGG                   310       320       330       340       350       360                       301                                                                               ATAAGCAGTC                                                                               TGTACGGAAT                                                                               TTATACAGAA                                                                               GCTTTTAGAG                                                                               AGTGGGAAGC                                                                               AGATCCTACT                361                                                                               AATCCAGCAT                                                                               TAAAAGAAGA                                                                               GATGCGTACT                                                                               CAATTTAATG                                                                               ACATGAACAG                                                                               TATTCTTGTA                421                                                                               ACAGCTATTC                                                                               CTCTTTTTTC                                                                               AGTTCAAAAT                                                                               TATCAAGTCC                                                                               CATTTTTATC                                                                               AGTATATGTT                481                                                                               CAAGCTGCAA                                                                               ATTTACATTT                                                                               ATCGGTTTTG                                                                               AGAGATGTTT                                                                               CAGTGTTTGG                                                                               GCAGGCTTGG                541                                                                               GGATTTGATA                                                                               TAGCAACAAT                                                                               AAATAGTCGT                                                                               TATAATGATC                                                                               TGACTAGACT                                                                               TATTCCTATA                   610       620       630       640       650       660                       601                                                                               TATACAGATT                                                                               ATGCTGTACG                                                                               CTGGTACAAT                                                                               ACGGGATTAG                                                                               ATCGCTTACC                                                                               ACGAACTGGT                661                                                                               GGGCTGCGAA                                                                               ACTGGGCAAG                                                                               ATTTAATCAG                                                                               TTTAGAAGAG                                                                               AGTTAACAAT                                                                               ATCAGTATTA                721                                                                               GATATTATTT                                                                               CTTTTTTCAG                                                                               AAATTACGAT                                                                               TCTAGATTAT                                                                               ATCCAATTCC                                                                               AACAAGCTCC                781                                                                               CAATTAACGC                                                                               GGGAAGTATA                                                                               TACAGATCCG                                                                               GTAATTAATA                                                                               TAACTGACTA                                                                               TAGAGTTGGC                841                                                                               CCCAGCTTCG                                                                               AGAATATTGA                                                                               GAACTCAGCC                                                                               ATTAGAAGCC                                                                               CCCACCTTAT                                                                               GGACTTCTTA                   910       920       930       940       950       960                       901                                                                               AATAATTTGA                                                                               CCATTGATAC                                                                               GGATTTGATT                                                                               AGAGGTGTTC                                                                               ACTATTGGGC                                                                               AGGGCATCGT                961                                                                               GTAACTTCTC                                                                               ATTTTACAGG                                                                               TAGTTCTCAA                                                                               GTGATAACAA                                                                               CCCCTCAATA                                                                               TGGGATAACC                1021                                                                              GCAAATGCGG                                                                               AACCAAGACG                                                                               AACTATTGCT                                                                               CCTAGTACTT                                                                               TTCCAGGTCT                                                                               TAACCTATTT                1081                                                                              TATAGAACAT                                                                               TATCAAATCC                                                                               TTTCTTCCGA                                                                               AGATCAGAAA                                                                               ATATTACTCC                                                                               TACCTTAGGG                1141                                                                              ATAAATGTAG                                                                               TACAGGGAGT                                                                               AGGGTTCATT                                                                               CAACCAAATA                                                                               ATGCTGAAGT                                                                               TCTATATAGA                   1210      1220      1230      1240      1250      1260                      1201                                                                              AGTAGGGGGA                                                                               CAGTAGATTC                                                                               TCTTAATGAG                                                                               TTACCAATTG                                                                               ATGGTGAGAA                                                                               TTCATTAGTT                1261                                                                              GGATATAGTC                                                                               ATCGATTAAG                                                                               TCATGTTACA                                                                               CTAACCAGGT                                                                               CGTTATATAA                                                                               TACTAATATA                1321                                                                              ACTAGCCTGC                                                                               CAACATTTGT                                                                               TTGGACACAT                                                                               CACAGTGCTA                                                                               CTAATACAAA                                                                               TACAATTAAT                1381                                                                              CCAGATATTA                                                                               TTACACAAAT                                                                               ACCTTTAGTG                                                                               AAAGGATTTA                                                                               GACTTGGTGG                                                                               TGGCACCTCT                1441                                                                              GTCATTAAAG                                                                               GACCAGGATT                                                                               TACAGGAGGG                                                                               GATATCCTTC                                                                               GAAGAAATAC                                                                               CATTGGTGAG                   1510      1520      1530      1540      1550      1560                      1501                                                                              TTTGTGTCTT                                                                               TACAAGTCAA                                                                               TATTAACTCA                                                                               CCAATTACCC                                                                               AAAGATACCG                                                                               TTTAAGATTT                1561                                                                              CGTTATGCTT                                                                               CCAGTAGGGA                                                                               TGCACGAATT                                                                               ACTGTAGCGA                                                                               TAGGAGGACA                                                                               AATTAGAGTA                1621                                                                              GATATGACCC                                                                               TTGAAAAAAC                                                                               CATGGAAATT                                                                               GGGGAGAGCT                                                                               TAACATCTAG                                                                               AACATTTAGC                1681                                                                              TATACCAATT                                                                               TTAGTAATCC                                                                               TTTTTCATTT                                                                               AGGGCTAATC                                                                               CAGATATAAT                                                                               TAGAATAGCT                1741                                                                              GAAGAACTTC                                                                               CTATTCGTGG                                                                               TGGTGAGCTT                                                                               TATATAGATA                                                                               AAATTGAACT                                                                               TATTCTAGCA                   1810      1820      1830      1840      1850      1860                      1801                                                                              GATGCAACAT                                                                               TTGAAGAAGA                                                                               ATATGATTTG                                                                               GAAAGAGCAC                                                                               AGAAGGCGGT                                                                               GAATGCCCTG                1861                                                                              TTTACTTCTA                                                                               CAAATCAACT                                                                               AGGGCTAAAA                                                                               ACAGATGTGA                                                                               CGGATTATCA                                                                               TATTGATCAA                1921                                                                              GTTTCCAATT                                                                               TAGTTGAGTG                                                                               TTTATCGGAT                                                                               GAATTTTGTC                                                                               TGGATGAAAA                                                                               GAGAGAATTA                1981                                                                              TCCGAGAAAG                                                                               TCAAACATGC                                                                               GAAGCGACTC                                                                               AGTGATGAAC                                                                               GGAATTTACT                                                                               TCAAGATCCA                2041                                                                              AACTTCAGAG                                                                               GGATCAATAG                                                                               GCAACCAGAC                                                                               CGTGGCTGGA                                                                               GAGGAAGCAC                                                                               GGATATTACT                   2110      2120      2130      2140      2150      2160                      2101                                                                              ATCCAAGGTG                                                                               GAGATGACGT                                                                               ATTCAAAGAG                                                                               AATTACGTCA                                                                               CATTACCGGG                                                                               TACCTTTGAT                2161                                                                              GAGTGCTATC                                                                               CAACGTATTT                                                                               ATATCAAAAA                                                                               ATAGATGAGT                                                                               CGAAGTTAAA                                                                               AGCTTATACC                2221                                                                              CGCTATGAAT                                                                               TAAGAGGGTA                                                                               TATCGAGGAT                                                                               AGTCAAGACT                                                                               TAGAAATCTA                                                                               TTTAATTCGC                2281                                                                              TACAATGCAA                                                                               AACACGAGAC                                                                               AGTAAACGTG                                                                               CCAGGTACGG                                                                               GTTCCTTATG                                                                               GCCGCTTTCA                2341                                                                              GCCCAAAGTC                                                                               CAATCGGAAA                                                                               GTGTGGAGAA                                                                               CCGAATCGAT                                                                               GCGCGCCACA                                                                               CCTTGAATGG                   2410      2420      2430      2440      2450      2460                      2401                                                                              AATCCTAATC                                                                               TAGATTGCTC                                                                               CTGCAGAGAC                                                                               GGGGAAAAAT                                                                               GTGCCCATCA                                                                               TTCCCATCAT                2461                                                                              TTCTCCTTGG                                                                               ACATTGATGT                                                                               TGGATGTACA                                                                               GACTTAAATG                                                                               AGGACTTAGG                                                                               TGTATGGGTG                2521                                                                              ATATTCAAGA                                                                               TTAAGACACA                                                                               AGATGGCTAT                                                                               GCAAGACTAG                                                                               GAAATCTAGA                                                                               GTTTCTCGAA                2581                                                                              GAGAAACCAC                                                                               TATTAGGGGA                                                                               AGCACTAGCT                                                                               CGTGTGAAAA                                                                               GAGCGGAGAA                                                                               AAAATGGAGA                2641                                                                              GACAAATGCG                                                                               AAAAATTGGA                                                                               ATGGGAAACA                                                                               AATATTGTTT                                                                               ATAAAGAGGC                                                                               AAAAGAATCT                   2710      2720      2730      2740      2750      2760                      2701                                                                              GTAGATGCTT                                                                               TATTTGTAAA                                                                               CTCTCAATAT                                                                               GATAGATTAC                                                                               AAGCGGATAC                                                                               GAATATCGCG                2761                                                                              ATGATTCATG                                                                               CGGCAGATAA                                                                               ACGCGTTCAT                                                                               AGCATTCGAG                                                                               AAGCGTATCT                                                                               GCCAGAGCTG                2821                                                                              TCTGTGATTC                                                                               CGGGTGTCAA                                                                               TGCGGCTATT                                                                               TTTGAAGAAT                                                                               TAGAAGGGCG                                                                               TATTTTCACT                2881                                                                              GCATTCTCCC                                                                               TATATGATGC                                                                               GAGAAATGTC                                                                               ATTAAAAATG                                                                               GCGATTTCAA                                                                               TAATGGCTTA                2941                                                                              TCATGCTGGA                                                                               ACGTGAAAGG                                                                               GCATGTAGAT                                                                               GTAGAAGAAC                                                                               AGAACAACCA                                                                               TCGTTCGGTC                   3010      3020      3030      3040      3050      3060                      3001                                                                              CTTGTTGTTC                                                                               CAGAATGGGA                                                                               AGCAGAAGTG                                                                               TCACAAGAAG                                                                               TTCGTGTTTG                                                                               TCCGGGTCGT                3061                                                                              GGCTATATCC                                                                               TTCGTGTTAC                                                                               AGCGTACAAA                                                                               GAGGGATATG                                                                               GAGAGGGCTG                                                                               TGTAACGATT                3121                                                                              CATGAGATCG                                                                               AAGACAATAC                                                                               AGACGAACTG                                                                               AAATTCAGCA                                                                               ACTGTGTAGA                                                                               AGAGGAAGTA                3181                                                                              TATCCAAACA                                                                               ACACGGTAAC                                                                               GTGTAATAAT                                                                               TATACTGCGA                                                                               CTCAAGAAGA                                                                               ACATGAGGGT                3241                                                                              ACGTACACTT                                                                               CCCGTAATCG                                                                               AGGATATGAC                                                                               GAAGCCTATG                                                                               AAAGCAATTC                                                                               TTCTGTACAT                   3310      3320      3330      3340      3350      3360                      3301                                                                              GCGTCAGTCT                                                                               ATGAAGAAAA                                                                               ATCGTATACA                                                                               GATAGACGAA                                                                               GAGAGAATCC                                                                               TTGTGAATCT                3361                                                                              AACAGAGGAT                                                                               ATGGGGATTA                                                                               CACACCACTA                                                                               CCAGCTGGCT                                                                               ATGTGACAAA                                                                               AGAATTAGAG                3421                                                                              TACTTCCCAG                                                                               AAACCGATAA                                                                               GGTATGGATT                                                                               GAGATCGGAG                                                                               AAACGGAAGG                                                                               AACATTCATC                3481                                                                              GTGGACAGCG                                                                               TGGAATTACT                                                                               TCTTATGGAG                                                                               GAATAATA                                      __________________________________________________________________________

                                      TABLE 2                                      __________________________________________________________________________     Deduced amino acid sequence of novel toxin.                                    __________________________________________________________________________                     5              10             15                                1  Met                                                                               Glu                                                                               Ile                                                                               Val                                                                               Asn                                                                               Asn                                                                               Gln                                                                               Asn                                                                               Gln                                                                               Cys                                                                               Val                                                                               Pro                                                                               Tyr                                                                               Asn                                                                               Cys                               16 Leu                                                                               Asn                                                                               Asn                                                                               Pro                                                                               Glu                                                                               Asn                                                                               Glu                                                                               Ile                                                                               Leu                                                                               Asp                                                                               Ile                                                                               Glu                                                                               Arg                                                                               Ser                                                                               Asn                               31 Ser                                                                               Thr                                                                               Val                                                                               Ala                                                                               Thr                                                                               Asn                                                                               Ile                                                                               Ala                                                                               Leu                                                                               Glu                                                                               Ile                                                                               Ser                                                                               Arg                                                                               Leu                                                                               Leu                               46 Ala                                                                               Ser                                                                               Ala                                                                               Thr                                                                               Pro                                                                               Ile                                                                               Gly                                                                               Gly                                                                               Ile                                                                               Leu                                                                               Leu                                                                               Gly                                                                               Leu                                                                               Phe                                                                               Asp                               61 Ala                                                                               Ile                                                                               Trp                                                                               Gly                                                                               Ser                                                                               Ile                                                                               Gly                                                                               Pro                                                                               Ser                                                                               Gln                                                                               Trp                                                                               Asp                                                                               Leu                                                                               Phe                                                                               Leu                               76 Glu                                                                               Gln                                                                               Ile                                                                               Glu                                                                               Leu                                                                               Leu                                                                               Ile                                                                               Asp                                                                               Gln                                                                               Lys                                                                               Ile                                                                               Glu                                                                               Glu                                                                               Phe                                                                               Ala                               91 Arg                                                                               Asn                                                                               Gln                                                                               Ala                                                                               Ile                                                                               Ser                                                                               Arg                                                                               Leu                                                                               Glu                                                                               Gly                                                                               Ile                                                                               Ser                                                                               Ser                                                                               Leu                                                                               Tyr                              106 Gly                                                                               Ile                                                                               Tyr                                                                               Thr                                                                               Glu                                                                               Ala                                                                               Phe                                                                               Arg                                                                               Glu                                                                               Trp                                                                               Glu                                                                               Ala                                                                               Asp                                                                               Pro                                                                               Thr                              121 Asn                                                                               Pro                                                                               Ala                                                                               Leu                                                                               Lys                                                                               Glu                                                                               Glu                                                                               Met                                                                               Arg                                                                               Thr                                                                               Gln                                                                               Phe                                                                               Asn                                                                               Asp                                                                               Met                              136 Asn                                                                               Ser                                                                               Ile                                                                               Leu                                                                               Val                                                                               Thr                                                                               Ala                                                                               Ile                                                                               Pro                                                                               Leu                                                                               Phe                                                                               Ser                                                                               Val                                                                               Gln                                                                               Asn                              151 Tyr                                                                               Gln                                                                               Val                                                                               Pro                                                                               Phe                                                                               Leu                                                                               Ser                                                                               Val                                                                               Tyr                                                                               Val                                                                               Gln                                                                               Ala                                                                               Ala                                                                               Asn                                                                               Leu                              166 His                                                                               Leu                                                                               Ser                                                                               Val                                                                               Leu                                                                               Arg                                                                               Asp                                                                               Val                                                                               Ser                                                                               Val                                                                               Phe                                                                               Gly                                                                               Gln                                                                               Ala                                                                               Trp                              181 Gly                                                                               Phe                                                                               Asp                                                                               Ile                                                                               Ala                                                                               Thr                                                                               Ile                                                                               Asn                                                                               Ser                                                                               Arg                                                                               Tyr                                                                               Asn                                                                               Asp                                                                               Leu                                                                               Thr                              196 Arg                                                                               Leu                                                                               Ile                                                                               Pro                                                                               Ile                                                                               Tyr                                                                               Thr                                                                               Asp                                                                               Tyr                                                                               Ala                                                                               Val                                                                               Arg                                                                               Trp                                                                               Tyr                                                                               Asn                              211 Thr                                                                               Gly                                                                               Leu                                                                               Asp                                                                               Arg                                                                               Leu                                                                               Pro                                                                               Arg                                                                               Thr                                                                               Gly                                                                               Gly                                                                               Leu                                                                               Arg                                                                               Asn                                                                               Trp                              226 Ala                                                                               Arg                                                                               Phe                                                                               Asn                                                                               Gln                                                                               Phe                                                                               Arg                                                                               Arg                                                                               Glu                                                                               Leu                                                                               Thr                                                                               Ile                                                                               Ser                                                                               Val                                                                               Leu                              241 Asp                                                                               Ile                                                                               Ile                                                                               Ser                                                                               Phe                                                                               Phe                                                                               Arg                                                                               Asn                                                                               Tyr                                                                               Asp                                                                               Ser                                                                               Arg                                                                               Leu                                                                               Tyr                                                                               Pro                              256 Ile                                                                               Pro                                                                               Thr                                                                               Ser                                                                               Ser                                                                               Gln                                                                               Leu                                                                               Thr                                                                               Arg                                                                               Glu                                                                               Val                                                                               Tyr                                                                               Thr                                                                               Asp                                                                               Pro                              271 Val                                                                               Ile                                                                               Asn                                                                               Ile                                                                               Thr                                                                               Asp                                                                               Tyr                                                                               Arg                                                                               Val                                                                               Gly                                                                               Pro                                                                               Ser                                                                               Phe                                                                               Glu                                                                               Asn                              286 Ile                                                                               Glu                                                                               Asn                                                                               Ser                                                                               Ala                                                                               Ile                                                                               Arg                                                                               Ser                                                                               Pro                                                                               His                                                                               Leu                                                                               Met                                                                               Asp                                                                               Phe                                                                               Leu                              301 Asn                                                                               Asn                                                                               Leu                                                                               Thr                                                                               Ile                                                                               Asp                                                                               Thr                                                                               Asp                                                                               Leu                                                                               Ile                                                                               Arg                                                                               Gly                                                                               Val                                                                               His                                                                               Tyr                              316 Trp                                                                               Ala                                                                               Gly                                                                               His                                                                               Arg                                                                               Val                                                                               Thr                                                                               Ser                                                                               His                                                                               Phe                                                                               Thr                                                                               Gly                                                                               Ser                                                                               Ser                                                                               Gln                              331 Val                                                                               Ile                                                                               Thr                                                                               Thr                                                                               Pro                                                                               Gln                                                                               Tyr                                                                               Gly                                                                               Ile                                                                               Thr                                                                               Ala                                                                               Asn                                                                               Ala                                                                               Glu                                                                               Pro                              346 Arg                                                                               Arg                                                                               Thr                                                                               Ile                                                                               Ala                                                                               Pro                                                                               Ser                                                                               Thr                                                                               Phe                                                                               Pro                                                                               Gly                                                                               Leu                                                                               Asn                                                                               Leu                                                                               Phe                              361 Tyr                                                                               Arg                                                                               Thr                                                                               Leu                                                                               Ser                                                                               Asn                                                                               Pro                                                                               Phe                                                                               Phe                                                                               Arg                                                                               Arg                                                                               Ser                                                                               Glu                                                                               Asn                                                                               Ile                              376 Thr                                                                               Pro                                                                               Thr                                                                               Leu                                                                               Gly                                                                               Ile                                                                               Asn                                                                               Val                                                                               Val                                                                               Gln                                                                               Gly                                                                               Val                                                                               Gly                                                                               Phe                                                                               Ile                              391 Gln                                                                               Pro                                                                               Asn                                                                               Asn                                                                               Ala                                                                               Glu                                                                               Val                                                                               Leu                                                                               Tyr                                                                               Arg                                                                               Ser                                                                               Arg                                                                               Gly                                                                               Thr                                                                               Val                              406 Asp                                                                               Ser                                                                               Leu                                                                               Asn                                                                               Glu                                                                               Leu                                                                               Pro                                                                               Ile                                                                               Asp                                                                               Gly                                                                               Glu                                                                               Asn                                                                               Ser                                                                               Leu                                                                               Val                              421 Gly                                                                               Tyr                                                                               Ser                                                                               His                                                                               Arg                                                                               Leu                                                                               Ser                                                                               His                                                                               Val                                                                               Thr                                                                               Leu                                                                               Thr                                                                               Arg                                                                               Ser                                                                               Leu                              436 Tyr                                                                               Asn                                                                               Thr                                                                               Asn                                                                               Ile                                                                               Thr                                                                               Ser                                                                               Leu                                                                               Pro                                                                               Thr                                                                               Phe                                                                               Val                                                                               Trp                                                                               Thr                                                                               His                              451 His                                                                               Ser                                                                               Ala                                                                               Thr                                                                               Asn                                                                               Thr                                                                               Asn                                                                               Thr                                                                               Ile                                                                               Asn                                                                               Pro                                                                               Asp                                                                               Ile                                                                               Ile                                                                               Thr                              466 Gln                                                                               Ile                                                                               Pro                                                                               Leu                                                                               Val                                                                               Lys                                                                               Gly                                                                               Phe                                                                               Arg                                                                               Leu                                                                               Gly                                                                               Gly                                                                               Gly                                                                               Thr                                                                               Ser                              481 Val                                                                               Ile                                                                               Lys                                                                               Gly                                                                               Pro                                                                               Gly                                                                               Phe                                                                               Thr                                                                               Gly                                                                               Gly                                                                               Asp                                                                               Ile                                                                               Leu                                                                               Arg                                                                               Arg                              496 Asn                                                                               Thr                                                                               Ile                                                                               Gly                                                                               Glu                                                                               Phe                                                                               Val                                                                               Ser                                                                               Leu                                                                               Gln                                                                               Val                                                                               Asn                                                                               Ile                                                                               Asn                                                                               Ser                              511 Pro                                                                               Ile                                                                               Thr                                                                               Gln                                                                               Arg                                                                               Tyr                                                                               Arg                                                                               Leu                                                                               Arg                                                                               Phe                                                                               Arg                                                                               Tyr                                                                               Ala                                                                               Ser                                                                               Ser                              526 Arg                                                                               Asp                                                                               Ala                                                                               Arg                                                                               Ile                                                                               Thr                                                                               Val                                                                               Ala                                                                               Ile                                                                               Gly                                                                               Gly                                                                               Gln                                                                               Ile                                                                               Arg                                                                               Val                              541 Asp                                                                               Met                                                                               Thr                                                                               Leu                                                                               Glu                                                                               Lys                                                                               Thr                                                                               Met                                                                               Glu                                                                               Ile                                                                               Gly                                                                               Glu                                                                               Ser                                                                               Leu                                                                               Thr                              556 Ser                                                                               Arg                                                                               Thr                                                                               Phe                                                                               Ser                                                                               Tyr                                                                               Thr                                                                               Asn                                                                               Phe                                                                               Ser                                                                               Asn                                                                               Pro                                                                               Phe                                                                               Ser                                                                               Phe                              571 Arg                                                                               Ala                                                                               Asn                                                                               Pro                                                                               Asp                                                                               Ile                                                                               Ile                                                                               Arg                                                                               Ile                                                                               Ala                                                                               Glu                                                                               Glu                                                                               Leu                                                                               Pro                                                                               Ile                              586 Arg                                                                               Gly                                                                               Gly                                                                               Glu                                                                               Leu                                                                               Tyr                                                                               Ile                                                                               Asp                                                                               Lys                                                                               Ile                                                                               Glu                                                                               Leu                                                                               Ile                                                                               Leu                                                                               Ala                              601 Asp                                                                               Ala                                                                               Thr                                                                               Phe                                                                               Glu                                                                               Glu                                                                               Glu                                                                               Tyr                                                                               Asp                                                                               Leu                                                                               Glu                                                                               Arg                                                                               Ala                                                                               Gln                                                                               Lys                              616 Ala                                                                               Val                                                                               Asn                                                                               Ala                                                                               Leu                                                                               Phe                                                                               Thr                                                                               Ser                                                                               Thr                                                                               Asn                                                                               Gln                                                                               Leu                                                                               Gly                                                                               Leu                                                                               Lys                              631 Thr                                                                               Asp                                                                               Val                                                                               Thr                                                                               Asp                                                                               Tyr                                                                               His                                                                               Ile                                                                               Asp                                                                               Gln                                                                               Val                                                                               Ser                                                                               Asn                                                                               Leu                                                                               Val                              646 Glu                                                                               Cys                                                                               Leu                                                                               Ser                                                                               Asp                                                                               Glu                                                                               Phe                                                                               Cys                                                                               Leu                                                                               Asp                                                                               Glu                                                                               Lys                                                                               Arg                                                                               Glu                                                                               Leu                              661 Ser                                                                               Glu                                                                               Lys                                                                               Val                                                                               Lys                                                                               His                                                                               Ala                                                                               Lys                                                                               Arg                                                                               Leu                                                                               Ser                                                                               Asp                                                                               Glu                                                                               Arg                                                                               Asn                              676 Leu                                                                               Leu                                                                               Gln                                                                               Asp                                                                               Pro                                                                               Asn                                                                               Phe                                                                               Arg                                                                               Gly                                                                               Ile                                                                               Asn                                                                               Arg                                                                               Gln                                                                               Pro                                                                               Asp                              691 Arg                                                                               Gly                                                                               Trp                                                                               Arg                                                                               Gly                                                                               Ser                                                                               Thr                                                                               Asp                                                                               Ile                                                                               Thr                                                                               Ile                                                                               Gln                                                                               Gly                                                                               Gly                                                                               Asp                              706 Asp                                                                               Val                                                                               Phe                                                                               Lys                                                                               Glu                                                                               Asn                                                                               Tyr                                                                               Val                                                                               Thr                                                                               Leu                                                                               Pro                                                                               Gly                                                                               Thr                                                                               Phe                                                                               Asp                              721 Glu                                                                               Cys                                                                               Tyr                                                                               Pro                                                                               Thr                                                                               Tyr                                                                               Leu                                                                               Tyr                                                                               Gln                                                                               Lys                                                                               Ile                                                                               Asp                                                                               Glu                                                                               Ser                                                                               Lys                              736 Leu                                                                               Lys                                                                               Ala                                                                               Tyr                                                                               Thr                                                                               Arg                                                                               Tyr                                                                               Glu                                                                               Leu                                                                               Arg                                                                               Gly                                                                               Tyr                                                                               Ile                                                                               Glu                                                                               Asp                              751 Ser                                                                               Gln                                                                               Asp                                                                               Leu                                                                               Glu                                                                               Ile                                                                               Tyr                                                                               Leu                                                                               Ile                                                                               Arg                                                                               Tyr                                                                               Asn                                                                               Ala                                                                               Lys                                                                               His                              766 Glu                                                                               Thr                                                                               Val                                                                               Asn                                                                               Val                                                                               Pro                                                                               Gly                                                                               Thr                                                                               Gly                                                                               Ser                                                                               Leu                                                                               Trp                                                                               Pro                                                                               Leu                                                                               Ser                              781 Ala                                                                               Gln                                                                               Ser                                                                               Pro                                                                               Ile                                                                               Gly                                                                               Lys                                                                               Cys                                                                               Gly                                                                               Glu                                                                               Pro                                                                               Asn                                                                               Arg                                                                               Cys                                                                               Ala                              796 Pro                                                                               His                                                                               Leu                                                                               Glu                                                                               Trp                                                                               Asn                                                                               Pro                                                                               Asn                                                                               Leu                                                                               Asp                                                                               Cys                                                                               Ser                                                                               Cys                                                                               Arg                                                                               Asp                              811 Gly                                                                               Glu                                                                               Lys                                                                               Cys                                                                               Ala                                                                               His                                                                               His                                                                               Ser                                                                               His                                                                               His                                                                               Phe                                                                               Ser                                                                               Leu                                                                               Asp                                                                               Ile                              826 Asp                                                                               Val                                                                               Gly                                                                               Cys                                                                               Thr                                                                               Asp                                                                               Leu                                                                               Asn                                                                               Glu                                                                               Asp                                                                               Leu                                                                               Gly                                                                               Val                                                                               Trp                                                                               Val                              841 Ile                                                                               Phe                                                                               Lys                                                                               Ile                                                                               Lys                                                                               Thr                                                                               Gln                                                                               Asp                                                                               Gly                                                                               Tyr                                                                               Ala                                                                               Arg                                                                               Leu                                                                               Gly                                                                               Asn                              856 Leu                                                                               Glu                                                                               Phe                                                                               Leu                                                                               Glu                                                                               Glu                                                                               Lys                                                                               Pro                                                                               Leu                                                                               Leu                                                                               Gly                                                                               Glu                                                                               Ala                                                                               Leu                                                                               Ala                              871 Arg                                                                               Val                                                                               Lys                                                                               Arg                                                                               Ala                                                                               Glu                                                                               Lys                                                                               Lys                                                                               Trp                                                                               Arg                                                                               Asp                                                                               Lys                                                                               Cys                                                                               Glu                                                                               Lys                              886 Leu                                                                               Glu                                                                               Trp                                                                               Glu                                                                               Thr                                                                               Asn                                                                               Ile                                                                               Val                                                                               Tyr                                                                               Lys                                                                               Glu                                                                               Ala                                                                               Lys                                                                               Glu                                                                               Ser                              901 Val                                                                               Asp                                                                               Ala                                                                               Leu                                                                               Phe                                                                               Val                                                                               Asn                                                                               Ser                                                                               Gln                                                                               Tyr                                                                               Asp                                                                               Arg                                                                               Leu                                                                               Gln                                                                               Ala                              916 Asp                                                                               Thr                                                                               Asn                                                                               Ile                                                                               Ala                                                                               Met                                                                               Ile                                                                               His                                                                               Ala                                                                               Ala                                                                               Asp                                                                               Lys                                                                               Arg                                                                               Val                                                                               His                              931 Ser                                                                               Ile                                                                               Arg                                                                               Glu                                                                               Ala                                                                               Tyr                                                                               Leu                                                                               Pro                                                                               Glu                                                                               Leu                                                                               Ser                                                                               Val                                                                               Ile                                                                               Pro                                                                               Gly                              946 Val                                                                               Asn                                                                               Ala                                                                               Ala                                                                               Ile                                                                               Phe                                                                               Glu                                                                               Glu                                                                               Leu                                                                               Glu                                                                               Gly                                                                               Arg                                                                               Ile                                                                               Phe                                                                               Thr                              961 Ala                                                                               Phe                                                                               Ser                                                                               Leu                                                                               Tyr                                                                               Asp                                                                               Ala                                                                               Arg                                                                               Asn                                                                               Val                                                                               Ile                                                                               Lys                                                                               Asn                                                                               Gly                                                                               Asp                              976 Phe                                                                               Asn                                                                               Asn                                                                               Gly                                                                               Leu                                                                               Ser                                                                               Cys                                                                               Trp                                                                               Asn                                                                               Val                                                                               Lys                                                                               Gly                                                                               His                                                                               Val                                                                               Asp                              991 Val                                                                               Glu                                                                               Glu                                                                               Gln                                                                               Asn                                                                               Asn                                                                               His                                                                               Arg                                                                               Ser                                                                               Val                                                                               Leu                                                                               Val                                                                               Val                                                                               Pro                                                                               Glu                              1006                                                                               Trp                                                                               Glu                                                                               Ala                                                                               Glu                                                                               Val                                                                               Ser                                                                               Gln                                                                               Glu                                                                               Val                                                                               Arg                                                                               Val                                                                               Cys                                                                               Pro                                                                               Gly                                                                               Arg                              1021                                                                               Gly                                                                               Tyr                                                                               Ile                                                                               Leu                                                                               Arg                                                                               Val                                                                               Thr                                                                               Ala                                                                               Tyr                                                                               Lys                                                                               Glu                                                                               Gly                                                                               Tyr                                                                               Gly                                                                               Glu                              1036                                                                               Gly                                                                               Cys                                                                               Val                                                                               Thr                                                                               Ile                                                                               His                                                                               Glu                                                                               Ile                                                                               Glu                                                                               Asp                                                                               Asn                                                                               Thr                                                                               Asp                                                                               Glu                                                                               Leu                              1051                                                                               Lys                                                                               Phe                                                                               Ser                                                                               Asn                                                                               Cys                                                                               Val                                                                               Glu                                                                               Glu                                                                               Glu                                                                               Val                                                                               Tyr                                                                               Pro                                                                               Asn                                                                               Asn                                                                               Thr                              1066                                                                               Val                                                                               Thr                                                                               Cys                                                                               Asn                                                                               Asn                                                                               Tyr                                                                               Thr                                                                               Ala                                                                               Thr                                                                               Gln                                                                               Glu                                                                               Glu                                                                               His                                                                               Glu                                                                               Gly                              1081                                                                               Thr                                                                               Tyr                                                                               Thr                                                                               Ser                                                                               Arg                                                                               Asn                                                                               Arg                                                                               Gly                                                                               Tyr                                                                               Asp                                                                               Glu                                                                               Ala                                                                               Tyr                                                                               Glu                                                                               Ser                              1096                                                                               Asn                                                                               Ser                                                                               Ser                                                                               Val                                                                               His                                                                               Ala                                                                               Ser                                                                               Val                                                                               Tyr                                                                               Glu                                                                               Glu                                                                               Lys                                                                               Ser                                                                               Tyr                                                                               Thr                              1111                                                                               Asp                                                                               Arg                                                                               Arg                                                                               Arg                                                                               Glu                           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                                                                     Thr                                                                               Pro                                                                               Leu                                                                               Pro                                                                               Ala                                                                               Gly                                                                               Tyr                                                                               Val                                                                               Thr                                                                               Lys                                                                               Glu                                                                               Leu                                                                               Glu                              1141                                                                               Tyr                                                                               Phe                                                                               Pro                                                                               Glu                                                                               Thr                                                                               Asp                                                                               Lys                                                                               Val                                                                               Trp                                                                               Ile                                                                               Glu                                                                               Ile                                                                               Gly                                                                               Glu                                                                               Thr                              1156                                                                               Glu                                                                               Gly                                                                               Thr                                                                               Phe                                                                               Ile                                                                               Val                                                                               Asp                                                                               Ser                                                                               Val                                                                               Glu                                                                               Leu                                                                               Leu                                                                               Leu                                                                               Met                                                                               Glu                              1171                                                                               Glu                                                                        __________________________________________________________________________

                                      TABLE 3                                      __________________________________________________________________________      ##STR1##                                                                       ##STR2##                                                                       ##STR3##                                                                       ##STR4##                                                                       ##STR5##                                                                       ##STR6##                                                                       ##STR7##                                                                       ##STR8##                                                                       ##STR9##                                                                       ##STR10##                                                                      ##STR11##                                                                      ##STR12##                                                                      ##STR13##                                                                      ##STR14##                                                                      ##STR15##                                                                      ##STR16##                                                                      ##STR17##                                                                      ##STR18##                                                                      ##STR19##                                                                      ##STR20##                                                                      ##STR21##                                                                      ##STR22##                                                                      ##STR23##                                                                      ##STR24##                                                                      ##STR25##                                                                      ##STR26##                                                                      ##STR27##                                                                      ##STR28##                                                                      ##STR29##                                                                      ##STR30##                                                                      ##STR31##                                                                      ##STR32##                                                                      ##STR33##                                                                      ##STR34##                                                                      ##STR35##                                                                      ##STR36##                                                                      ##STR37##                                                                      ##STR38##                                                                      ##STR39##                                                                      ##STR40##                                                                      ##STR41##                                                                      ##STR42##                                                                      ##STR43##                                                                      ##STR44##                                                                      ##STR45##                                                                      ##STR46##                                                                      ##STR47##                                                                      ##STR48##                                                                      ##STR49##                                                                      ##STR50##                                                                      ##STR51##                                                                      ##STR52##                                                                      ##STR53##                                                                      ##STR54##                                                                      ##STR55##                                                                      ##STR56##                                                                      ##STR57##                                                                      ##STR58##                                                                      ##STR59##                                                                     __________________________________________________________________________

    TABLE 4       HD1 -- WHITELEY'S "4.5" GENE HD73 -- ADANG'S "6.6" GENE BTB -- BULLA'S      "5.3" GENE 81F -- MYCOGEN'S 81F TOX GENE BTE -- HONEE'S ENTOMOCIDUS TOX      GENE HD2 -- BRIZZARD'S HD2 TOX GENE         1    5     10     15     20     25     30  |    |        |     |     |     |     |      HD1 M D N N P N I N E C I P Y N C L S N P E V E V L G G E R I E HD73 --      -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --      -- -- -- -- -- BTB -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --      -- -- -- -- -- -- -- -- -- -- -- -- 81F -- E I V N -- Q -- Q -- V -- --      -- -- -- N -- -- -- N -- I -- D I -- -- S N BTE -- E E N N Q = -- Q --      -- -- -- -- -- -- -- -- -- -- E V L -- D --  -- -- -- S HD2 -- T S N R*      -- E -- -- I -- N A V S N H S A Q M D L -- P D A -- -- --      * K      35     40     45     50     55 56    60      |     |        |     |     | |    | HD1      T G Y T P I D I S L S L T Q F L L S E F V P G A G F V L G L HD73 -- --      -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --  -- -- -- --      -- -- -- -- BTB -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --      -- -- -- -- -- -- -- -- -- -- -- 81F S T = V A T N -- A -- E I S R = --      -- A S A T -- I G -- I L -- -- -- BTE -- -- N S S -- -- -- -- -- -- -- V      -- -- -- V -- N -- -- -- -- G -- -- L V -- -- HD2 D S* S T V Q T G I N I      A G R I -- G V L V -- -- F --  -- *Y S F = -- * L C I S E G N N I D P F      V S A * Q L A S F       65     70     75     80     85     90      |     |     |     |     |        | HD1 V D I I W G I F G P S Q W D A F P V Q I E Q L I N Q R I      E E HD73 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- L --  -- -- --      -- -- -- -- -- -- -- -- -- BTB -- -- -- -- -- -- -- -- -- -- -- -- -- --      -- -- L -- -- -- -- -- -- -- -- -- -- -- -- -- 81F F -- A -- -- -- S I      -- -- -- -- -- -- L -- L E -- -- -- L -- -- D -- K -- -- -- BTE I -- F V      -- -- -- V -- -- -- -- -- -- -- -- L -- -- -- -- -- -- -- -- E -- -- G      -- HD2 -- G E L -- P R = -- R D -- -- E I -- L E H V -- -- -- -- -- -- Q      -- T --       95     100     105     110 111    115     120      |     |     |     | |      |     | HD1 F A R N Q A I S R L E G L S N L Y Q I Y A      E S F R E W E A D HD73 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --      -- -- -- -- -- -- -- -- -- -- -- -- -- -- BTB --  -- -- -- -- -- -- --      -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- 81F --      -- -- -- -- -- -- -- -- -- -- -- I -- S -- -- G -- -- T -- A -- -- -- --      -- -- -- BTE -- -- -- -- A -- -- A N -- -- -- -- G -- N F N -- -- V -- A      -- K -- -- -- E -- HD2 N -- -- -- T -- L A -- -- Q -- -- G D S F R A --      Q Q -- L E D -- L E N       125     130     135     140     145     150          |     |     |     |     .vertline      .     | HD1 P T N P A L R E E M R I Q F N D M N S A L T T A I P      L L A V HD73 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --      -- -- -- -- -- -- -- F -- -- BTB -- -- -- -- -- -- -- -- -- -- -- -- --      -- -- -- -- -- -- -- -- -- -- -- -- -- -- F -- -- 81F -- -- -- -- -- --      K -- -- -- -- T -- -- -- -- -- -- -- I -- V -- -- -- -- -- F S -- BTE --      N -- -- E T -- T R V I D R -- R I L D G L -- E R D -- -- S F R I HD2 R D      D A R T -- S V L Y T -- Y I A L E L D F L N -- M -- -- F -- I       155         160     165 166    170     175     180      |     |          | |    |     |     |      HD1 Q N Y Q V P L L S V Y V Q A A N L H L S V L R D V S V F G Q HD73 --      -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --      -- -- -- -- -- BTB -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --      -- -- -- -- -- -- -- -- -- -- -- -- 81F -- -- -- -- -- -- F -- -- --  --      -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- BTE S G F E --      -- -- -- -- -- -- A -- -- -- -- -- -- V A I -- -- -- S V I -- -- E HD2 R      -- Q E -- -- -- -- M -- -- A -- -- -- -- -- -- -- L L -- -- -- A -- L --      -- S       185     190     195     200     205     210      |        |     |     |     |     |      HD1 R W G F D A A T I N S R Y N D L T R L I G N Y T D Y A V R W HD73 --      -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --      -- -- -- -- -- BTB -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --      -- -- -- -- -- -- -- H -- -- -- -- 81F A -- -- -- -- I -- -- -- -- -- --      -- -- -- -- -- -- -- --  P I -- -- -- -- -- -- -- -- BTE -- -- -- L T T      I N V -- E N -- -- R -- I -- H -- D E -- A -- H C A N T HD2 E F -- L T S      Q E -- Q = -- -- Y E = = -- Q V E* D -- S -- -- C -- E -- * R T R      215     220 221    225     230     235     240      |      | |    |     |     |      | HD1 Y N T G L E R V = W G P D S R D W V R Y N Q F R R E L T L      T HD73 -- -- -- -- -- -- -- -- = -- -- -- -- -- -- -- -- -- -- -- -- --      -- -- -- -- -- -- -- -- BTB -- -- -- -- -- -- -- -- = -- -- -- -- -- --      -- -- I -- -- -- -- -- -- -- -- -- -- -- -- 81F -- -- -- -- -- D -- L P      R T G G L -- N -- A -- F -- -- -- -- -- -- -- -- I S BTE -- -- R -- -- N      N L = = K -- T Y Q -- -- I T -- -- R L -- -- D -- -- -- -- HD2 -- -- --      -- -- N S L = R -- T N A A S -- -- -- -- -- -- -- -- -- D -- -- -- G        245     250     255     260     265     270      |      |     |     |     |     |      HD1 V L D I V A L F S N Y D S R R Y P I R T V S Q L T R E I Y T HD73 --      -- -- -- -- -- -- -- P -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --      -- -- -- -- -- BTB -- -- -- -- -- S -- -- P -- -- -- -- -- T -- -- -- --      -- -- -- -- -- -- -- -- -- -- -- 81F -- -- -- -- I S F -- R -- -- -- --      -- L -- -- -- P -- S -- -- -- -- -- -- V -- -- BTE -- -- -- -- A -- F --      P -- --  -- N -- -- -- -- -- Q P -- G -- -- -- -- -- V -- -- HD2 -- --      -- L -- -- -- -- P S -- -- T -- T -- -- -- N -- S A -- -- -- -- -- V --      --       275 276    280     285     290     295     300      |      |    |     |     |     |       | HD1 N P V L E N F D G S F R = G M A = Q R I E Q N = I R Q P      H L HD73 -- -- -- -- --  -- -- -- -- -- -- -- = -- S -- = -- G -- -- R S      -- -- -- S -- -- -- BTB -- -- -- -- -- -- -- -- -- -- -- -- = -- S -- =      -- G -- -- G S -- -- -- S -- -- -- 81F D -- -- I N I T -- = = Y -- V --      P S F E N -- -- N S A -- -- S -- -- -- BTE D -- L I = -- -- N P Q L Q =      S V -- = --* N V M E S R* I R N P H L * L P T F * R HD2 D  A I* Y N N N      A P -- -- S A I E A = = = = = = A A -- -- S -- -- -- * G A T G V N M A S      M N W       305     310     315     320     325     330      |         |     |     |     |     |       HD1 M D I L N S I T I Y T D V H R G F N Y W S G H Q I T A S P V HD73 --      -- -- -- -- -- -- -- -- -- -- -- A -- -- -- Y Y -- -- -- -- -- -- -- M      -- -- -- -- BTB -- -- -- -- -- -- -- -- -- -- -- -- A -- -- -- E Y -- --      -- -- -- -- -- M -- -- -- -- 81F -- -- F -- -- N L -- -- D -- -- L I --      -- V H -- -- A -- -- R V -- S H = = BTE F -- -- -- -- N L -- -- F -- --*      -- G -- N -- = -- -- G -- -- R V I C -- L I * W F S HD2 L -- F -- E Q L      -- -- F S A S S --* H M T -- -- R -- -- T -- Q S R -- I * W S N T R      331    335     340     345     350     355     360  |      |     |     |     |     |        | HD1 G F S G P E = = F A F P L F G N A G N A A P P = = V L V      S = HD73 -- -- -- -- -- -- = = -- T -- -- -- Y -- T M -- -- -- -- -- Q Q      R I V A Q = BTB -- -- -- -- -- -- = = -- T -- -- -- Y -- T M -- -- -- --      -- Q Q R I V A Q = 81F = -- T -- S S Q V I T T -- Q Y -- I T A -- -- E      -- R = = R T I A P BTE -- = = -- G N =  = I T S -- I Y -- R E A -- Q E A      -- = = R S F T = HD2 -- = G -- = = = = L N T S T H -- A T N T S I N -- =      = -- T L R =       365     370     375     380     385 386    390      |     |     |     |     |      |    | HD1 = = L T G L G I F = R T L S S P L Y R R I =      = = = = I L G S HD73 = = -- = -- Q -- V Y = -- -- -- -- -- T -- -- -- --      P = = = = = = F N I BTB = = -- = -- Q -- V Y = -- -- -- -- -- T -- -- --      -- P = = = = = = F N I 81F S T F P -- -- N L -- Y -- -- -- -- N -- F F      -- -- S E N I T P T -- -- I BTE = = F N -- = P V -- = -- -- -- -- N -- T      L -- = = = = = = L L Q Q P HD2 = = F A S R D V Y = -- --  E -- = = = --      A G V = = = = = L -- = W       395     400     405     410     415      420      |     |     |     |      |     | HD1 G P N N Q E L F V L D G T E F S F A S L T      T N L P S T I Y R HD73 -- I -- -- -- Q -- S -- -- -- -- -- -- -- A Y G T      -- = S -- -- -- -- A V -- -- BTB -- I -- -- -- Q -- S -- -- -- -- -- --      -- A Y G T = S S -- -- -- -- A V -- -- 81F N = = = = = = = -- V Q -- V G      -- = = = = I Q P -- N A E V L -- -- BTE W -- A P R = F N L R G V E G V E      -- S T P -- N S F T = = = -- -- HD2 -- I Y L E P I H G V P T V R -- N --      T N = = = = = -- Q N I = S       425     430     435     440 441    445         450      |     |     |     |      |    |     | HD1 Q R G T V D S L D V I P P Q      D N S V P P R A G F S H R L S H HD73 K S -- -- -- -- -- -- -- E -- -- --      -- N -- N -- -- -- -- Q -- -- -- -- -- -- -- -- BTB K S -- -- -- -- --      -- -- E -- -- -- -- N -- N -- -- -- -- Q -- -- -- -- -- -- -- -- 81F S      -- -- -- -- -- -- -- N E L -- I D G E N = = S L V -- Y -- -- -- -- -- --      BTE G -- -- -- -- -- -- -- T E L -- -- E -- -- -- -- -- -- -- E -- Y --      -- -- -- C -- HD2 D -- -- -- A N* S E T E L -- -- E T T E R -- N Y E S Y      -- -- -- -- -- -- * Y S Q P Y E S P G L Q L K D       455     460      465     470     475     480      |     |     |           |     |     | HD1 V T M L S Q A = = A G      A V Y T L R A P T F S W Q H R S A E F HD73 -- S -- F R S G F S N S S --      S I I -- -- -- M -- -- -- I -- -- -- -- -- -- BTB -- S -- F R S G F S N      S S -- S I I -- -- -- M -- -- -- I -- -- -- -- -- -- 81F -- -- = -- T R      S L Y N T N I T S -- = = -- -- -- V -- T -- H -- -- T N BTE A -- F V =      -- R = = S -- T P F L T T G V V -- -- -- T D -- -- -- T L HD2 I G I I L      -- S = = R V N -- = = = = = -- V Y -- -- T -- -- -- -- D R       485      490     495 496    500     505     510      |     |       | |    |     |     | HD1 N      N I I P S S Q I T Q I P L T K S T N L G S G T S V V K G P HD73 -- -- --      -- A -- D S -- -- -- -- -- A V -- G N F -- F N -- = -- -- I S -- -- BTB      -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --      -- -- -- -- -- -- 81F T -- T -- N P D I -- -- -- -- -- -- V -- G F R --      -- G -- -- -- -- I -- -- -- BTE T -- T -- D P E R -- N -- -- -- -- V --      G F R V W G -- -- -- -- I T -- -- HD2 T --  T -- G P N R -- -- -- -- --      M V -- A S E -- P Q -- -- T -- -- R -- --       515     520     525      530     535     540      |     |     |      |     |     | HD1 G F T G G D I L R R T S P G      Q I S T L R V N I T A P L = = = HD73 -- -- -- -- -- -- L V -- L N -- S      -- N N I Q N -- G Y -- E V -- I H F P BTB -- -- -- -- -- -- -- -- -- --      -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- = = = 81F -- -- -- --      -- -- -- -- -- -- N T I -- E F V S -- Q -- -- -- N S -- I = = = BTE --      -- -- -- -- -- -- -- -- -- N T F -- D F V S -- Q -- -- -- N S -- I = = =      HD2 -- -- -- -- -- -- -- -- -- -- -- N T -- G F G P I -- -- T V N G --      -- = = =       545     550 551    555     560     565     570      |     | |    |     |      |     | HD1 = = S Q R Y R V R I R Y A S T T N L Q F H      T S I D G R P I N HD73 S T -- T -- -- -- -- -- V -- -- -- -- V -- P I H      L N V N W G N S S -- = BTB = = -- -- -- -- -- -- -- -- -- -- -- -- -- --      -- -- -- -- -- -- -- -- -- -- -- -- -- -- 81F = = T -- -- -- -- L -- F      -- -- -- -- S R D A R I T V A -- G -- Q I R V BTE = = T -- -- -- -- L --      F -- -- -- -- S R D A R V I V L T G V G G Q* V * V S HD2 = = T -- -- --      -- I G F -- -- -- -- -- V D F D -- F V -- R G -- T T V --       575      580     585     590     595     600      |     |      |     |     |     | HD1 Q G N F S A      T M S S G S = = = = N L Q S G S F R T V G F = = HD73 = = = -- -- N -- V      P A T A T S L D -- -- -- -- S D -- = = = G Y F E BTB -- -- -- -- -- --      -- -- -- -- -- -- = = = = -- -- -- -- -- -- -- -- -- -- -- -- = = 81F D      M T L E K -- -- E I -- E = = = = S -- T -- R T -- S Y T N -- = = BTE N M      P L Q K -- -- E I -- E = = = = -- -- T -- R T -- -- Y T D -- = = HD2 N F      R -- L R -- -- N -- -- D = = = = E -- K Y -- N -- V R R A -- = =      605 606    610     615     620     625     630      | |          |     |     |     |     .vertline      . HD1 = T T P F N F S N G S S V F T L S A H V F N S G N E V Y I D HD73 S      A N A -- T S -- L -- N I -- = = = G V R N -- S G T A G -- I -- -- BTB =      -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --      -- -- -- -- -- 81F = S N -- -- S -- R A N P D I I R I A E E L P E R -- G      -- L -- -- -- BTE = S N P F S -- R A N P D I I G I -- E Q P L F G A G*      -- L -- -- -- * S I S S G HD2 = -- -- -- -- T F T Q I Q D I I R T -- I Q G       L S G N G -- -- -- -- --       635     640     645     650     655      660      |     |     |     |      |     | HD1 R I E F V P A E V T F E A E Y D L E R A Q      K A V N E L F T S HD73 -- F -- -- I -- V T A -- L -- -- -- -- N -- -- --      -- -- -- -- -- -- A -- -- -- -- BTB -- -- -- -- -- -- -- -- -- -- -- --      -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- 81F K -- -- L I L      -- D A -- -- -- E -- -- -- -- -- -- -- -- -- -- -- -- A -- -- -- -- BTE      K -- -- I I L -- D A -- -- -- -- -- S -- -- -- -- -- -- -- -- -- -- A --      -- -- -- HD2 K -- -- I I -- V T A -- -- -- -- -- -- -- -- -- -- -- -- E      -- -- -- A -- -- -- N   661    665     670     675     680     685      690  |    |     |     |     .vertline      .     |     | HD1 S N Q I G L K T D V T D Y H I D Q V      S N L V E C L S D E F C HD73 T -- -- L -- -- -- -- N -- -- -- -- -- --      -- -- -- -- -- -- --  T Y -- -- -- -- -- -- BTB -- -- -- -- -- -- -- --      -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- 81F T      -- -- L -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --      -- -- -- -- -- BTE -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --      -- -- -- -- D -- -- -- -- -- -- -- HD2 T -- P R R -- -- -- -- -- -- --      -- -- -- -- -- -- -- -- -- -- A -- -- -- -- -- -- --       695     700        705     710     715 716    720      |     |      |     |     | |    | HD1 L      D E K Q E L S E K V K H A K R L S D E R N L L Q D P N F R HD73 -- -- --      -- R -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- S --      -- K BTB -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --      -- -- -- -- -- -- -- -- -- 81F -- -- -- -- R -- -- -- -- -- -- -- -- --      -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- BTE -- -- -- -- R -- --      -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- HD2      -- -- -- -- R -- -- L -- -- -- -- Y -- -- -- -- -- -- -- -- -- -- -- --      -- -- -- -- T       725     730     735     740     745     750      |     |     |     |     |        | HD1 G I N R Q L D R G W R G S T D I T I Q G G D D V F K E N      Y V HD73 D -- -- -- -- P E -- -- -- G -- -- -- G -- -- -- -- -- -- -- --      -- -- -- -- -- -- -- BTB -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --      -- -- -- -- -- -- -- -- -- -- -- -- -- -- 81F -- -- -- -- -- P -- -- --      -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- BTE -- --      -- -- -- R -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --      -- -- -- -- HD2 S -- -- K -- P D* H -- --  W -- -- E N -- -- -- -- E --      N -- -- -- -- -- -- -- -- * F I S T N E Q S N F T S I H E Q S E      755     760     765     770 771    775     780      |      |     |     | |    |      | HD1 T L L G T F D E C Y P T Y L Y Q K I D E S K L K A Y T R Y      Q HD73 -- -- S -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --      -- -- -- F -- -- -- -- BTB -- -- -- -- -- -- -- -- -- -- -- -- -- -- --      -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- 81F -- -- P -- -- -- -- --      -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- E BTE --      -- P -- -- V -- -- -- -- A -- -- -- -- -- -- --  -- -- -- -- -- -- -- --      -- -- -- E HD2 -- -- P -- -- -- N -- -- -- -- -- -- -- -- -- -- -- G --      -- E -- -- -- -- -- -- -- --       785     790     795     800     805        810      |     |     |     |      |     | HD1 L R G Y I E D S Q D L E I Y L I R Y N A K      H E T V N V P G T HD73 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --      -- -- -- -- -- -- -- -- -- -- -- -- -- -- BTB -- -- -- -- -- -- -- -- --      -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- 81F -- --      -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --      -- -- -- -- BTE -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --      -- -- -- -- -- I -- -- -- -- -- -- HD2 -- -- -- -- -- -- -- -- -- -- --      -- -- -- -- -- -- -- -- -- -- -- -- -- L D -- -- -- --       815     820          825 826    830     835     840      |     |      | |    |     |     | HD1 G      S L W P L S A Q S P I G K C G E P N R C A P H L E W N P D HD73 -- -- --      -- -- -- -- --  -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --      -- -- -- BTB -- -- -- -- -- -- -- -- P -- -- -- -- -- -- = = = = = = = =      = = = = = = = 81F -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --      -- -- -- -- -- -- -- -- -- -- -- N BTE -- -- -- -- -- -- -- -- -- -- --      -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- HD2 E -- -- --      -- -- -- V E -- -- -- -- R -- -- -- -- -- -- -- -- -- -- F -- -- -- --      --       845     850     855     860     865     870      |      |     |     |     |     |      HD1 L D C S C R D G E K C A H H S H H F S L D I D V G C T D L N HD73 --      -- -- -- -- -- -- -- --  -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --      -- -- -- -- -- BTB = = = = = = = = = = = -- -- -- -- -- -- -- -- -- --      -- -- -- -- -- -- -- -- -- 81F -- -- -- -- -- -- -- -- -- -- -- -- -- --      -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- BTE V -- -- -- -- -- --      -- -- -- -- -- -- -- -- -- -- --  T -- -- -- -- -- -- -- -- -- -- -- HD2      -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --      -- -- -- -- -- H       875     880 881    885     890     895     900        |     | |    |     |      |     | HD1 E D L G V W V I F K I K T Q D G H A R L G      N L E F L E E K P HD73 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --      -- -- -- -- -- -- -- -- -- -- -- -- -- -- BTB -- -- -- -- -- -- -- -- --      -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- 81F -- --      -- -- -- -- -- -- -- -- -- -- -- -- -- -- Y -- -- -- -- -- -- -- -- --      -- -- -- -- BTE -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --      -- -- -- -- -- -- -- -- -- -- -- HD2 -- N -- -- -- -- -- V -- -- -- --      -- -- E -- -- -- -- -- -- -- -- -- -- I -- -- -- --       905     910       915     920     925     930      |     |     .vertline      .     |     |     | HD1 L V G E A L A R V K R      A E K K W R D K R E K L E W E T N I V HD73 -- -- -- -- -- -- -- -- -- --      -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- BTB -- -- --      -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --      -- -- -- 81F -- L -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- C      -- -- -- -- -- -- -- -- -- -- BTE -- L -- -- -- -- -- -- -- -- -- -- --      -- -- -- -- -- -- -- -- -- -- Q L -- -- -- -- -- HD2 -- L -- -- -- -- S      -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- Q L -- -- K R --      935 936    940     945     950     955     960      | |          |     |     |     |     .vertline      . HD1 Y K E A K E S V D A L F V N S Q Y D Q L Q A D T N I A M I H HD73      -- -- -- -- -- -- -- -- -- --  -- -- -- -- -- -- -- -- -- -- -- -- -- --      -- -- -- -- -- -- BTB -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --      -- R -- -- -- -- -- -- -- -- -- -- -- 81F -- -- -- -- -- -- -- -- -- --      -- -- -- -- -- -- -- -- R -- -- -- -- -- -- -- -- -- -- -- BTE -- -- --      -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- R --  -- V -- -- -- -- --      -- -- -- HD2 -- T -- -- -- -- A -- -- -- -- -- -- D -- -- -- -- R -- --      -- -- -- -- -- G -- -- --       965     970     975     980     985      990      |     |     |     |      |     | HD1 A A D K R V H S I R E A Y L P E L S V I P      G V N A A I F E E HD73 -- -- -- -- -- -- -- -- -- -- -- --  -- -- -- --      -- -- -- -- -- -- -- -- -- -- -- -- -- -- BTB -- -- -- -- -- -- -- -- --      -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- 81F -- --      -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --      -- -- -- -- BTE -- -- -- -- -- -- -- R -- -- -- -- -- -- -- -- -- -- --      -- --  -- -- -- -- -- -- -- -- -- HD2 -- -- -- -- L -- -- R -- -- -- --      -- -- S -- -- P -- -- -- -- -- -- -- E -- -- -- --   991    995     1000          1005     1010     1015     1020  |    |      |     |     |     |     |      HD1 L E G R I F T A F S L Y D A R N V I K N G D F N N G L S C W HD73 --      -- -- -- -- -- -- -- -- -- -- --  -- -- -- -- -- -- -- -- -- -- -- -- --      -- -- -- -- -- BTB -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --      -- -- -- -- -- -- -- -- -- -- -- -- 81F -- -- -- -- -- -- -- -- -- -- --      -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- BTE -- -- -- --      -- -- -- -- Y -- -- -- -- -- -- -- -- -- -- -- --  -- -- -- -- -- -- L      -- -- HD2 -- -- -- H -- I -- -- I -- -- -- -- -- -- -- -- V -- -- -- --      -- -- -- -- -- T -- --       1025     1030     1035     1040     1045      1046    1050      |     |     |     |           | |    | HD1 N V K G H V D V E E Q N N Q      R S V L V L P E W E A E V S Q E HD73 -- -- -- -- -- -- -- -- -- -- -- --      -- -- -- -- -- -- -- V -- -- -- -- -- -- -- -- -- -- BTB -- -- -- -- --      -- -- -- -- -- -- -- -- H -- -- -- -- -- V -- -- -- -- -- -- -- -- -- --      81F -- -- -- -- -- -- -- -- -- -- -- -- -- H -- -- -- -- -- V -- -- --      -- -- -- -- -- -- -- BTE -- -- -- -- -- -- -- -- -- -- -- -- -- H -- --      -- -- -- I -- -- -- -- -- -- -- -- -- -- HD2 -- -- -- -- -- -- -- -- Q =      -- S H H -- -- D -- -- I -- -- -- -- -- -- -- -- -- A       1055      1060     1065     1070     1075     1080      |     |         |     |     |     | HD1 V R V C P      G R G Y I L R V T A Y K E G Y G E G C V T I H E I HD73 -- -- -- -- -- --      -- -- -- -- -- -- -- -- -- -- --  -- -- -- -- -- -- -- -- -- -- -- -- --      BTB -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --      -- -- -- -- -- -- -- 81F -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --      -- -- -- -- -- -- -- -- -- -- -- -- -- -- BTE -- -- -- -- -- -- -- -- --      -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- HD2 -- --      -- -- -- -- C -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --      -- -- -- --       1085     1090     1095     1100 1101    1105     1110          |     |     |     | |       |     | HD1 E N N T D E L K F S N C V E E E I Y P N N      T V T C N D Y T V HD73 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --      -- -- -- -- -- -- -- -- -- -- -- -- -- -- BTB -- -- -- -- -- -- -- -- --      -- -- -- -- -- -- -- V -- -- -- -- -- -- -- -- -- -- -- -- A 81F -- D --      -- -- -- -- -- -- -- -- -- -- -- -- -- V -- -- -- -- -- -- -- -- -- N --      -- A BTE -- D -- -- -- -- -- -- -- -- -- -- -- -- -- -- V -- -- -- -- --      -- -- -- -- N -- -- G HD2 -- -- -- -- -- -- -- -- -- K -- R E -- -- -- V      -- -- T D -- G -- -- -- -- -- -- A       1115     1120     1125     1130          1135     1140      |     |     |      |     |     | HD1 N Q E E Y G G A Y T S R N R      G Y N E A = = = = P S V P A D Y HD73 -- -- -- -- -- -- -- -- -- -- -- --      -- -- -- -- -- -- --  = = = = -- -- -- -- -- -- -- BTB T -- -- -- -- E      -- T -- -- -- -- -- -- -- -- D G -- Y E S N S -- -- -- -- -- -- 81F T --      -- -- H E -- T -- -- -- -- -- -- -- -- D -- -- Y = = = E -- N S S V H      BTE T -- -- -- -- E -- T -- -- -- -- -- Q -- -- D -- -- Y G N N R -- --      -- -- -- -- HD2 H -- G T A --* D -- C N -- -- -- A -- -- E D -- Y E V D      T T A S V N -- * C A       1145     1150     1155 1156    1160     1165         1170      |     |     | |      |     |     | HD1 A S V Y E E K S Y T D G R R       N P C E F N R G Y R D Y T P L HD73 -- -- -- -- -- -- -- -- -- -- -- --      -- --  -- -- -- -- -- -- -- -- -- --  -- -- -- -- -- BTB -- -- A -- --      -- -- A -- -- -- -- -- --  -- -- -- -- S -- -- -- -- G -- -- -- -- --      81F -- -- -- -- -- -- -- -- -- -- -- R -- --  -- -- -- -- S -- -- -- --      G -- -- -- -- -- BTE -- -- -- -- -- -- -- -- -- -- -- -- -- --  -- -- --      -- S -- -- -- -- G -- -- -- -- -- HD2 K P T -- -- -- E T -- -- -- V --      --  -- H -- -- Y D -- -- -- V N Y P -- V       1175     1180     1185       1190     1195      |     |     |      |     | HD1 P V G Y V T K E L E Y F P E T D K V W I E      I G E T E G T F I HD73 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --      -- -- -- -- -- -- -- -- -- -- -- -- -- -- BTB -- A -- -- -- -- -- -- --      -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- 81F -- A      -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --      -- -- -- -- BTE -- A -- -- -- -- -- D -- -- -- -- -- -- -- -- -- -- --      -- -- -- -- -- -- -- -- -- -- -- HD2 -- A -- -- -- -- -- -- -- -- -- --      -- -- -- -- T -- -- -- -- -- -- -- -- -- -- K -- --  HD1 V D S V E L L L      M E HD73 -- -- -- -- -- -- -- -- -- -- BTB -- -- -- -- -- -- -- -- -- --      81F -- -- -- -- -- -- -- -- -- -- BTE -- -- -- -- -- -- -- -- -- -- HD2      -- -- -- -- -- -- -- -- -- --      HD1 is the cryA1 toxin gene from Bacillus thuringiensis subsp. kurstaki      HD1 (Brizzard and Whiteley, Nucleic acids Research 16(1988)2723.      HD73 is the cryA3 gene from HD73.      BTB is the cryA2 gene from BT strain Berliner.      81F is a delta endotoxin gene from Mycogen's BT strain PS81F.      BTE is a delta endotoxin gene from BT subspecies entomocidus (Honee, Salm      and Visser, Nucleic Acids Research 16(1988)6240.      HD2 is a delta endotoxin gene from BT strain HD2 (Brizzard and Whitely,      Nucleic Acids Research 16(1988)2723.      -- denote identical amino acid homologies.      = denote gaps required to align sequences with HD1.      *denote inserts required to align the sequences BTE and HD2 with HD1. 

We claim:
 1. A process for controlling lepidopteran insect pests which comprises contacting said insect pests with an insect-controlling effective amount of B. thuringiensis PS81F having all the identifying characteristics of NRRL B-18424.
 2. The process, according to claim 1, wherein said insect pests belong to the order Lepidoptera.
 3. The process, according to claim 2, wherein said insect pest is the Western spruce budworm.
 4. The process, according to claim 1, wherein said insect pest is contacted with an insect-controlling effective amount of B. thuringiensis PS81F, by incorporating said B. thuringiensis PSS81F into a bait granule and placing said granule on or in the soil when planting seed of a plant upon which plant insect pest is known to feed.
 5. A process for controlling soil-inhabiting insect pests of the order Lepidoptera which comprises(1) preparing a bait granule comprising B. thuringiensis PS81F, spores or crystals; and (2) placing said bait granule on or in the soil.
 6. The process, according to claim 5, wherein said bait granule is applied at the same time corn seed is planted in the soil.
 7. The process, according to claim 1 or 5, wherein substantially intact B.t. PS81F cells are treated to prolong the pesticidal activity when the substantially intact cells are applied to the environment of a target pest.
 8. A composition of matter comprising B. thuringiensis PS81F, spores or crystals in association with an insecticide carrier.
 9. The composition of matter, according to claim 8, wherein said carrier comprises phagostimulants or attractants.
 10. A composition of matter comprising B. thuringiensis PS81F in association with formulation ingredients applied as a seed coating.
 11. Bacillus thuringiensis PS81F, having all the identifying characteristics of NRRL B-128424 having activity against insect pests of the order Lepidoptera. 